For over a century since the discovery of X-rays by Rventgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. With the funding support of the parent R01, we have shown recently that 1) reactive oxygen species mediate this process;2) targeted cytoplasmic irradiation results in lipid peroxidation of membranes as shown by the induction of 4-hdyroxy-2-nonenal, a major lipid peroxidation byproduct;3) generated mitochondrial DNA depleted (Ao) AL cells and human fibroblasts;and finally, targeted cytoplasmic irradiation induces bystander mutations and chromatid breaks among neighboring, non-irradiate cells through a gap junction-mediated process. This raised the following questions: What are the effects of targeted cytoplasmic irradiation on mitochondrial DNA mutations and does mitochondrial DNA depleted human fibroblasts demonstrated reduced induction of mutagenesis upon cytoplasmic irradiation? The central hypotheses for this one year supplemental application is targeted cytoplasmic irradiation induces a dose dependent mutagenesis of mitochondrial DNA in human fibroblasts and that mitochondrial DNA depleted cells respond poorly to genotoxic signaling. To address these hypotheses, 2 specific aims are proposed. Mutations will be scored at the HPRT locus in human fibroblasts and mitochondrial DNA mutations (depletion, heteroplasmic deletions and point mutations will be determined. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. PUBLIC HEALTH RELEVANCE: Generations of students in radiation biology have been taught that heritable biological effects induced by ionizing radiation are the consequence of a direct radiation-nuclear interaction. Using the Columbia University charged particle microbeam and the human hamster hybrid (AL) cell mutagenic assay, there is evidence that targeted cytoplasmic irradiation is mutagenic in mammalian cells. This first, unequivocal demonstration of an extranuclear effect of ionizing radiation provides strong support of the subsequent, broad reaching bystander/ non-targeted effects of radiation. There is evidence that targeted cytoplasmic irradiation can also induce a bystander effect. As such, a better understanding of the genotoxic mechanism of cytoplasmic irradiation is critical in low dose radiation risk assessment. Mitochondria are widely distributed in the cytosol and mitochondrial DNA is highly susceptible to oxidative damage. The objective of the proposed study is to ascertain the possible effects of cytoplasmic irradiation on mitochondrial DNA mutations and the subsequent modulation on mitochondrial function. The study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.